Cationic cyclic amine and amphipathic transfection reagents

ABSTRACT

Cationic cyclic amine containing polymers and copolymers as well as novel lipids have been designed and synthesized for efficient delivery of nucleic acids to cells in biological systems, specifically for in vitro cell transfection research.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/632,385, filed Feb. 26, 2015, now U.S. Pat. No. 10,619,162, thecontents of which are all hereby incorporated by reference.

FIELD OF THE INVENTION

The field of the present invention is compounds comprising cationiccyclic amine containing copolymers, novel lipids and the use of suchreagents for delivering nucleic acids to a cell.

BACKGROUND

The present invention relates to cationic polymer and lipid compoundswhich have use in the delivery of nucleic acid to cells in biologicalsystems, for instance in in vitro cell transfection research. Theinvention also relates to methods of making such compounds andpotentially to gene therapy using such compounds.

The control of living processes is mediated through nucleic acids.Nucleic acids encode proteins which, as enzymes, hormones and otherregulatory factors, carry out the processes which enable livingorganisms to function. Nucleic acids also encode for regulatorysequences which control the expression of proteins.

Because of its central role in living organisms, nucleic acids make anideal therapeutic target. It is thought that many diseases could becontrolled by the manipulation of nucleic acids in living organisms.

The key factor limiting therapies based on nucleic acid manipulation isthe ability to deliver nucleic acids to the appropriate compartment ofthe cells. Nucleic acids are fragile molecules which are highlynegatively charged (one negative charge per phosphate group) and whichare readily cleaved by nucleases present both in extracellular fluidsand intracellular compartments. As a highly charged molecule it will notcross the lipid membranes surrounding the cell, nor can it readilyescape from endosomal compartments involved in the uptake ofmacromolecules into cells. Even RNAi molecules, although smaller inmolecular weight, show significant problems of stability and uptake.

The efficient delivery of biologically active compounds to theintracellular space of cells has been accomplished by the use of a widevariety of vesicles. One particular type of vesicle, liposomes, is oneof the most developed types of vesicles for drug delivery. Liposomes,which have been under development since the 1970's, are microscopicvesicles that comprise amphipathic molecules which contain bothhydrophobic and hydrophilic regions. Liposomes can be formed from onetype of amphipathic molecule or several different amphipathic molecules.Several methods have been developed to complex biologically activecompounds with liposomes. In particular, polynucleotides complexed withliposomes have been delivered to mammalian cells. After publication ofDOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), anumber of cationic lipids have been synthesized for this purpose.Essentially all the cationic lipids are amphipathic compounds thatcontain a hydrophobic domain, a spacer, and positively-charged amine(s).The cationic lipids are sometimes mixed with a fusogenic lipid such asDOPE (dioleoyl phosphatidyl ethanolamine) to form liposomes. Thecationic liposomes are then mixed with plasmid DNA and the binarycomplex of the DNA and liposomes are applied to cells in a tissueculture dish or injected in vivo. The ease of mixing the plasmid DNAwith the cationic liposome formulation, the ability of the cationiclipids to complex with DNA and the relative high levels of transfectionefficiency has led to increasing use of these formulations. However,these cationic lipid formulations have a common deficiency in that theyare typically toxic to the cells in culture and in vivo. More recentlylipids have been used in association with other DNA-binding compounds tofacilitate cell transfection.

The use of cationic polymers overcomes some, but not all, of theproblems associated with cationic lipid formulations. Polycationicpolymers are, however, generally cytotoxic although some cationicpolymers with lower toxicity have been reported. Cationic polymers aregenerally cheap to produce, and do not have the shelf life problemsassociated with cationic lipids.

Cationic polymers are very efficient at condensing nucleic acids into asmall volume and at protecting nucleic acids from degradation by serumnucleases. Interaction is through an equilibrium reaction in whichadjustment of the environmental conditions, (salt concentration, pH,molecular weight of each of the polymers) will affect the compositionand form of the complexes.

In the formation of toroids, the processes of condensation of nucleicacids and aggregation of particles are competing, so that these systemstend to be unstable with time and form larger aggregates. This isinfluenced by the charge ratio of the complexes, and can be reduced byusing an excess of one of the components. Generally such complexes are,therefore, made with an excess of polymer and/or lipid, although similarcomplexes with an excess of nucleic acids also have some favorableproperties.

Cationic polyamines such as polyethylenimine (PEI), poly(L-lysine),polyamidoamines, chitosan, poly(amino ester)s and polyacrylates havebeen widely investigated as nucleic acid delivery vehicles.

In comparison to cationic polymers containing aliphatic amino moieties,cyclic amine containing polymers has received significantly less or noattention in terms of their development delivery polymers. The cyclicamine moieties exhibit different physical and chemical propertiescompared to their aliphatic or aromatic counterparts

It is an object of the invention to overcome at least some of the aboveproblems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the general structure of cationic cyclic amine containingpolymers and copolymers.

FIG. 2 shows the general structure of cationic cyclic amine containingacrylamide/acrylate and alkyl acrylamide/acrylate copolymers.

FIG. 3 shows the general method for synthesizing tert-Butyloxycarbonyl(BOC) protected cyclic amine containing monomers.

FIG. 4 shows RAFT polymerization of BOC protected monomers in thepresence of a chain transfer agent (CTA), free radical initiator (Init),solvent (e.g. butyl acetate, BuAc), and heat (60-100 C).

FIG. 5 shows the removal of BOC protecting groups to form cationicpolymers with cyclic amine moieties.

FIG. 6 shows the ¹H NMR of (S)—N—BOC-pyrrolidinyl acrylate in CDCl₃.

FIG. 7 shows the ¹H NMR of poly((S)—N—BOC-pyrrolidinyl acrylate) inCDCl₃.

FIG. 8 shows the ¹H NMR of poly((S)-pyrrolidinyl acrylate) in D₂O.

FIG. 9 shows the general experimental for piperazine [andhomopiperazine] based compounds (n=1, 2; R₁ & R₂=hydrocarbon; R₃ &R₄=—C(═O)—R5).

FIG. 10 shows amide bond formation.

FIG. 11 shows data from an experiment comparing the claimed cyclicamines and standard lipids with a competitor's transfection reagent andthe same lipids.

FIG. 12 shows data from an experiment comparing the claimed lipid withmultiple competitor reagents using cell line A and cell line B.

FIG. 13 shows representative R₃ and R₄ groups of the lipid transfectionreagent.

FIG. 14 shows representative R₆ groups of the lipid transfectionreagent.

SUMMARY

The invention pertains to the use of synthetic cationic cyclic aminecontaining polymers and copolymers as nucleic acid transfection agents.In some instances, the polycations described are used in conjunctionwith novel endosomolytic lipids to transfect nucleic acids into cells.

The repeat units of the polycations described here have one or morecyclic amines and can be either secondary or tertiary, or a combinationthereof. In one embodiment, the precursor hydroxide or amine unit, andthe acrylate or acrylamide monomer containing secondary and/or tertiarycyclic amine side groups are protected with tert-butoxycarbonyl (BOC)protecting moieties. The BOC protected precursors can be used to formhomopolymers or various types of copolymers by grafting to a formedmacromolecule via the hydroxy or amino functional group. Alternatively,the BOC protected monomers can be used to form homopolymers or varioustypes of copolymers with acrylamide and/or acrylate co-monomers. The BOCgroups are subsequently removed under acidic conditionspost-modification or post-polymerization to form the requiredpolycations.

The development, synthesis, and characterization of cationic cyclicamine copolymers are described. Various cyclic amine copolymerscontaining tertiary, secondary, and/or primary amines were synthesizedusing free radical polymerization. Specifically, reversible-additionfragmentation chain transfer (RAFT) polymerization was used tosynthesize polyacrylamides with well-defined structures, compositions,and molecular weights (Mw/Mn<1.5). Architectures include, but are notlimited to, random/statistical, gradient, block, linear, branched,cross-linked/network, star, and dendritic structures.

The present invention provides a compound to assist nucleic acidtransfer into animal cells via a complex comprising nucleic acid and acationic cyclic amine containing polymers and copolymers. A novelcompound and method of preparation thereof, is described.

In a preferred embodiment, compositions comprising nucleic acids andcationic cyclic amine containing polymers and copolymers and/or lipids,and processes using such compositions to deliver a nucleic acid to ananimal cell in vivo or in vitro for the purposes of altering expressionof a gene in the cell are described.

In a preferred embodiment, compositions and compounds are described thatfacilitate delivery of nucleic acid to an animal cell in vitro and invivo. The nucleic acid comprises a double stranded structure having anucleotide sequence substantially identical to part of an expressedtarget nucleic acid within the cell. Further, the use of a cationiccyclic amine containing polymers and copolymers and/or lipidssignificantly increased nucleic acid transfer efficiency. The nucleicacid then alters expression of a selected endogenous nucleic acid.

In a preferred embodiment, the cationic cyclic amine containing polymersand copolymers and/or lipids is used to assist transfection of DNA, RNA,mRNA or RNAi into a cell. The nucleic acid then alters the cell'snatural process.

RNA interference (RNAi) is a phenomenon wherein double-stranded RNA,when present in a cell, inhibits expression of a gene that has anidentical or nearly identical sequence. Inhibition is caused bydegradation of the messenger RNA (mRNA) transcribed from the targetgene. The double-stranded RNA responsible for inducing RNAi is termedinterfering RNA. dsRNA introduced into the cytoplasm of a cell is firstprocessed into RNA fragments 21-25 nucleotides long. It has been shownin in vitro studies that these dsRNAs, termed small interfering RNAs(siRNA) are generated at least in part by the RNAse III-like enzymeDicer. Each siRNA is unwound into two single-stranded (ss) ssRNAs, thepassenger strand and the guide strand. The passenger strand is degraded,and the guide strand is incorporated into the RNA-induced silencingcomplex (RISC). The most studied outcome is post-transcriptional genesilencing, which occurs when the guide strand base pairs with acomplementary sequence in a messenger RNA molecule and induces cleavageby Argonaute, the catalytic component of the RISC complex.

RNAi has become a valuable research tool, both in cell culture and inliving organisms, because synthetic dsRNA introduced into cells canselectively and robustly induce suppression of specific genes ofinterest. RNAi may be used for large-scale screens that systematicallyshut down each gene in the cell, which can help identify the componentsnecessary for a particular cellular process or an event such as celldivision. The pathway is also used as a practical tool in biotechnologyand medicine. The cationic polyacrylamides described in thisspecification provide a mechanism to transfect siRNA and other nucleicacids into cells.

The development, synthesis, and characterization of cationic cyclicamine containing polymers and copolymers polymers are described. Variouscyclic amine containing polymers and copolymers were synthesized usingfree radical polymerization. Specifically, reversible-additionfragmentation chain transfer (RAFT) polymerization was used tosynthesize polyacrylamides with well-defined structures, compositions,and molecular weights (Mw/Mn<1.5). Architectures include, but are notlimited to, random/statistical, gradient, block, linear, branched,cross-linked/network, star, and dendritic structures. RAFT has rivaledother controlled free radical polymerization techniques such as atomtransfer radical polymerization (ATRP) as one of the most effective waysto synthesize well-defined and novel polymers. The controlled synthesisof RAFT polymers is achieved using conventional radical initiators suchas azobisisobutyronitrile (AIBN), and the reversible chain transfer ofdithiocarbonyl compounds.

Polymers: A polymer is a molecule built up by repetitive bondingtogether of smaller units called monomers. In this application the termpolymer includes both oligomers which have two to about 80 monomers andpolymers having more than 80 monomers. The polymer can be linear,branched network, star, comb, or ladder types of polymer. The polymercan be a homopolymer in which a single monomer is used or can becopolymer in which two or more monomers are used. Types of copolymersinclude alternating, random, block and graft. The main chain of apolymer is composed of the atoms whose bonds are required forpropagation of polymer length. The side chain of a polymer is composedof the atoms whose bonds are not required for propagation of polymerlength. To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process.

Steric Stabilizer: A steric stabilizer is a long chain hydrophilic groupthat prevents aggregation of final polymer by sterically hinderingparticle to particle electrostatic interactions. Examples include: alkylgroups, PEG chains, polysaccharides, alkyl amines. Electrostaticinteractions are the non-covalent association of two or more substancesdue to attractive forces between positive and negative charges.

Buffers: Buffers are made from a weak acid or weak base and their salts.Buffer solutions resist changes in pH when additional acid or base isadded to the solution.

Biochemical reactions: Biological, chemical, or biochemical reactionsinvolve the formation or cleavage of ionic and/or covalent bonds.

Reactive: A compound is reactive if it is capable of forming either anionic or a covalent bond with another compound. The portions of reactivecompounds that are capable of forming covalent bonds are referred to asreactive functional groups.

Steroid: A steroid derivative means a sterol, a sterol in which thehydroxyl moiety has been modified (for example, acylated), or a steroidhormone, or an analog thereof. The modification can include spacergroups, linkers, or reactive groups.

Sterics: Steric hindrance, or sterics, is the prevention or retardationof a chemical reaction because of neighboring groups on the samemolecule.

EXAMPLES

Polymers with a (meth)acrylate or (meth)acrylamide backbone and a cyclicamine containing side group (three or more carbons) containing one ormore secondary or tertiary amines are described (FIG. 1 ). Thecopolymers can be a combination of two or more different cationic repeatunit structures, or can be a combination of (meth)acrylamide and(meth)acrylate cationic units (FIG. 1 ). Copolymers can be a combinationof (meth)acrylate or (meth)acrylamide cyclic amine containing cationicunits and alkyl (meth)acrylate or (meth)acrylamide units (FIG. 2 ).

FIG. 3 highlights the tert-Butyloxycarbonyl (BOC) protected cyclic aminoacrylate and acrylamide monomers synthesized and polymerized by MinisBio LLC. The monomers are synthesized by reacting acryloyl chloride ormethacryloyl chloride with either hydroxyl groups or amines (primary orsecondary) in the presence of a base (usually diisopropylethylamine) andsolvent (usually dichloromethane). Structure and purity of the monomersis determined by ¹H NMR (FIG. 6 ). These monomers then undergopolymerization and copolymerization (FIG. 4 ). In this instance, theRAFT polymerization process is shown. Once the (co)polymers are purifiedby precipitation (usually into hexane), they are analyzed by gelpermeation chromatography (organic solvent phase) and ¹H NMR (FIG. 7 ).The (co)polymers are deprotected under acidic conditions to remove theBOC protecting groups (FIG. 5 ); structure and purity confirmed by ¹HNMR (FIG. 8 ).

All BOC protected cyclic amine containing monomers were synthesizedbased on the reaction of either acryloyl chloride or methacryloylchloride with a primary amine containing moiety in the presence of abase (FIG. 3 ). The synthesis of 1-(N—BOC-piperidyl)-4-acrylamide(14PipAm) is described here as an example:

4-amino-1-boc-piperidine (5.00 g, 0.025 mol) was dissolved indichloromethane (50 mL) and added to a dry 250 mL 3 neck round bottomflask flushed with nitrogen and equipped with a dropping funnel andstirrer bar. The flask was immersed in an ice bath before acryloylchloride (2.47 g, 0.0272 mol) in dichloromethane (15 mL) was added tothe stirring solution drop-wise via the dropping funnel over a period of45 min. The solution was stirred overnight and allowed to warm to roomtemperature. The solution was washed with 10% w/v citric acid solution(20 mL), 10% potassium carbonate solution (20 mL), saturated sodiumbicarbonate solution (20 mL), and brine (20 mL). The organic layer wasthen dried over sodium sulfate and passed through a basic alumina plug.The solvent was then removed by rotor evaporation at room temperature.The oil product was dissolved in dichloromethane (10 mL) and precipitatethree times into hexane. If necessary, a silica column is also used topurify the monomer. Yield=4.0 g (63%). ¹H NMR, δ(CDCl₃) ppm: 1.32 (2H),1.45 (9H), 1.95 (2H), 2.87 (2H), 4.03 (3H), 5.52 (1H), 5.66 (1H), 6.10(1H), 6.30 (1H).

Example 1

Polymer synthesis: The monomers described were polymerized using RAFT inorder to synthesize polymers of well-defined molecular weights,compositions, and architectures. The synthesis ofpoly(1-piperidyl)-4-acrylamide) (P14PipAm) is given as an example.

1-(N—BOC-piperidyl)-4-acrylamide (0.200 g, 0.787 mmol),4-cyano-4(phenylcarbonothioylthio)pentanoic acid (CPCPA, 1.12 mg,0.00401 mmol), AIBN (0.098 mg, 0.00060 mmol), and butyl acetate (1.00mL) were added to a 20 mL glass vial with stirrer bar. The vial wassealed with a rubber cap and the solution bubbled with nitrogen using along syringe with a second syringe as the outlet for 1 h. The syringeswere removed and the vial heated to 80° C. for 15 h using an oil bath.The solution was allowed to cool to room temperature and precipitatedinto hexane. The product was re-dissolved in dichloromethane andprecipitated into hexane dried under reduced pressure for several hours.Yield=0.181 mg (90%). ¹H NMR, δ(CDCl₃) ppm: 1.4 (9H), 1.8 (3H), 2.2(2H), 2.8 (2H), 3.8 (2H), 4.0 (3H).

The BOC protected polymers were deprotected post-polymerization to yieldprimary and secondary amines in the polymer side groups. Thedeprotection of P14PipAm-BOC is described as an example.

P14PipAm-BOC (0.150 g) was dissolved in a 2N HCl solution of aceticacidic (4 mL) and stirred for 1 h. Water (15 mL) was added to thesolution, which was then dialyzed against salt water and then deionizedwater over a period of 48 h. The dialyzed solution was then frozen andlyophilized to dryness (Yield=0.080 g).

Example 2

Lipid synthesis: To a cooled solution of 1,4-bis(aminoalkyl)piperazine(1 eq) and Et3N (2.1 eq) in CHCl3 is added, dropwise, a solution of acylchloride (2.05-2.1 eq) in CHCl3. The reaction mixture is stirred at roomtemperature overnight. The reaction mixture is diluted with CHCl3 todouble the volume, washed three times with saturated Na2CO3, washed oncewith saturated NaCl, and dried with MgSO4 or Na2SO4. The solvent isremoved using a rotary evaporator. The bisamide is purified byrecrystallization or by column chromatography.

To a stirred suspension of lithium aluminum hydride (LAH, 3 eq) intetrahydrofuran (THF), under nitrogen, is added, dropwise, a solution ofbisamide (1 eq) in THF. When the addition is complete, the reactionmixture is refluxed under nitrogen overnight. Then the reaction mixtureis cooled (cold water bath), and excess hydride is decomposed followingstandard procedure(s). The mixture is filtered, and the precipitate iswashed with THF. The filtrate is diluted with CHCl3 to at least doublethe volume, washed twice with water, washed once with saturated NaCl,and dried with MgSO4 or Na2SO4. The solvents are removed using a rotaryevaporator. The amine is purified by column chromatography. (see FIG. 9)

(Bis-His-ODAP) To a solution of 155.1 mg (2.211×10-4 mol) of ODAP in 6mL of THF was added 80.9 μL (4.645×10-4 mol) of DIEA, followed by 210.1mg (4.644×10-4 mol) of Boc-His(1-Boc)-OSu. The reaction mixture wasstirred at room temperature overnight (20 hr). Then the reaction mixturewas diluted with 50 mL of CHCl3, washed with saturated Na2CO3 (3×25 mL),washed with water (25 mL), washed with saturated NaCl (25 mL), driedwith MgSO4, and evaporated. Column chromatography on silica gel withCHCl3/MeOH=93:7 afforded 203.7 mg (67%) of Bis-(Boc-His(1-Boc))-ODAP asan oil: Rf=0.26 (CHCl3/MeOH=93:7), 0.49 (CHCl3/MeOH=90:10) (I2 orKMnO4); 1H NMR (400 MHz, CDCl3, TMS) δ 7.97 (s, 2H), 7.14 (s, 2H),5.4-5.3 (m, 6H, alkene-H+NH), 4.9-4.8 (m, 2H), 3.6-3.3 (m, 4H), 3.3-3.0(m, 4H), 3.0-2.7 (m, 4H), 2.6-2.2 (m, 8H), 2.3-2.2 (m, 4H), 2.1-2.0 (m,8H), 1.8-1.6 (m, 4H), 1.59 (s, 18H), 1.5-1.4 (m, 4H), 1.39 (s, 18H),1.4-1.2 (m, 44H), 0.88 (t, J=6.8 Hz, 6H); MS (ESI) m/z 1376.1 (M+),1276.0 ([M-Boc]+), 688.6 (M+2), 638.6 ([M-Boc]+2).

To a solution of 196.5 mg (1.428×10-4 mol) of Bis-(Boc-His(1-Boc))-ODAPin 12 mL of THF was added 6 mL of 6N HCl. After stirring at roomtemperature overnight, the reaction mixture was rotovapped. The crudeHCl salt was purified by column chromatography on silica gel withCHCl3/MeOH/NH4OH=85:15:2 to afford 115.0 mg (83%) of Bis-His-ODAP as acolorless oil: Rf=0.09 (CHCl3/MeOH/NH4OH=85:15:2), (ninhydrin, I2 orKMnO4); 1H NMR (400 MHz, CDCl3, TMS) δ5.4-5.3 (m, 4H), 4.1-4.0 (m, 2H,αH), 3.5-3.1 (m, 8H), 3.0-2.7 (m, 4H) 2.5-2.1 (m, 12H), 2.1-2.0 (m, 8H),1.8-1.6 (m, 4H), 1.6-1.4 (m, 4H), 1.4-1.2 (m, 44H), 0.88 (t, J=7.0 Hz,6H); MS (MALDI) m/z 975.77 (M+).

(Bis-(N-MeHis)-ODAP) To a solution of 437.8 mg (8.556×10-4 mol) ofBoc-N-Me-His(Trt)-OH and 260.9 mg (3.730×10-4 mol) of ODAP was added327.9 μL (1.883 mmol) of DIEA, and then 261.5 mg (1.027 mmol) of BOP—Cl.After stirring at room temperature for 1.5 hr, the reaction mixture waspartitioned between 50 mL of CHCl3 and 25 mL of water. The CHCl3 phasewas dried with MgSO4, and evaporated. Column chromatograpy on silica gelwith 5% MeOH in CHCl3 afforded 586.4 mg (93%) ofBis-(Boc-N-Me-His(Trt))-ODAP as an oil: Rf=0.25 (CHCl3/MeOH=95:5) (UV,I2); 1H NMR (400 MHz, CDCl3, TMS) δ 7.4-7.3 (m, 20H), 7.2-7.1 (m, 12H),6.57 (br s, 2H), 5.4-5.3 (m, 5H), 5.2-5.1 (m, 1H), 3.6-3.3 (m, 4H),3.3-3.1 (m, 4H), 3.1-2.8 (m, 4H), 2.77 (s, 3H), 2.74 (s, 3H), 2.6-2.2(m, 8H), 2.3-2.2 (m, 4H), 2.1-2.0 (m, 8H), 1.8-1.6 (m, 4H), 1.5-1.4 (m,4H), 1.37 (s, 18H), 1.4-1.2 (m, 44H), 0.88 (t, J=7.0 Hz, 6H).

To a 100 mL rb flask containing 580.5 mg (3.438×10-4 mol) ofBis-(Boc-N-Me-His(Trt))-ODAP was added 13 mL of 95% TFA. The materialwas dissolved, and stirred at room temperature. After 45 min, thesolvent was evaporated, and the residue was dried under vacuum. Columnchromatography on silica gel with CHCl3/MeOH/NH4OH=90:10:1 then 85:15:1afforded 243.1 mg (70%) of Bis-(N-MeHis)-ODAP as an oil: Rf=0.13(CHCl3/MeOH/NH4OH=90:10:1), 0.33 (CHCl3/MeOH/NH4OH=85:15:1) (I2); 1H NMR(400 MHz, CDCl3, TMS) δ 7.6-7.5 (m, 2H). 6.9-6.8 (m, 2H), 5.4-5.3 (m,4H), 5.1-5.0 (m, 2H), 3.7-3.5 (m, 4H), 3.5-3.2 (m, 4H), 3.0-2.7 (m, 4H),2.6-2.2 (m, 12H), 2.36 (s, 3H), 2.34 (m, 3H), 2.1-2.0 (m, 8H), 1.8-1.6(m, 4H), 1.6-1.4 (m, 4H), 1.4-1.2 (m, 44H), 0.88 (t, J=6.8 Hz, 6H); MS(MALDI) m/z 1003.9547 (M+), 1021.9617 ([M+H2O]+). (see FIG. 10 )

Example 3

Transfection efficiency of cationic cyclic amine polymers andBis-δ-Ava-ODAP or bis-(linoleyl)-4P4 relative to a commerciallyavailable reagent.

All transfections were performed in triplicate in 96 well plates usingsuspension 293-F cells grown in serum-free complete media. Cells wereseeded at 500,000 cells/mL at time of transfection. Transfectioncompetent complexes were prepared by first mixing 0.1 ug (per well)plasmid DNA (pCIluc-luciferase expression plasmid) with 10 μl Opti-MEMreduced serum media, followed by sequential addition of the polycation(cationic cyclic amines 3103 and 3301) and amphipathic compound (1112 or1180). Ternary complexes were incubated for 20 minutes before drop-wiseaddition to cultured cells. Cultured cells were grown in 100 μLserum-free complete media. No media change is requiredpost-transfection.

In this example cationic cyclic amines 3103 or 3301 served as thepolycation and Bis-δ-Ava-ODAP (1112) or Bis-(linoleyl)-4P4 (1180) servedas the amphipathic compound. Comparisons were made with the commerciallyavailable reagent jetPEI® (Polyplus Transfection) according to themanufacturer's recommended protocol (FIG. 11 ). Cells were harvested at36 hours and assayed for luciferase activity. FIG. 11 depicts the meanrelative light units for each experimental condition. The error barsrepresent the standard deviation of the triplicate wells.

At optimal dose and ratio, the combination of cationic cyclic amine 3103and amphipathic 1180 resulted up to a 4-fold increase in relative lightunits versus commercially available reagents. This demonstrates thetransfection efficiency of cationic cyclic amine 3103+Bis-(linoleyl)-4P4when complexed with pDNA and transfected into cells in culture.

TABLE 1 Structure of cationic polymer side chains Polymer NomenclatureR₁ R₂ R₃ X Y Monomer Structure 1 R2PA H (CH₂)₂ CH₂ O CH

2 S2PA H (CH₂)₂ CH₂ O CH

3 14PipA H (CH₂)₂ (CH₂)₂ O CH

4 14PipMA CH₃ (CH₂)₂ (CH₂)₂ O CH

5 R1PipA H (CH₂)₃ CH₂ O CH

6 S1PipA H (CH₂)₃ CH₂ O CH

7 PipzA H (CH₂)₂ (CH₂)₂ — N

8 PipEtA H (CH₂)₂ (CH₂)₂ O (CH₂)₂N

9 PipPrA H (CH₂)₂ (CH₂)₂ O (CH₂)₃N

10 PipAm H (CH₂)₂ (CH₂)₂ NH CH

11 PipMeAm H (CH₂)₂ (CH₂)₂ NH (CH₂)₂

12 PipMAm CH₃ (CH₂)₂ (CH₂)₂ NH CH

13 HHAzAm H (CH₂)₃ (CH₂)₂ NH CH

Example 4

Transfection efficiency of EPEI and Bis-(N-MeHis)-ODAP (1161) relativeto commercially available reagents.

All transfections were performed in triplicate in 96 well plates using(A) primary Human Umbilical Vein Endothelial Cells (HUVEC) or (B) JAWSII cells. Cells were approximately 70% confluent at time oftransfection. Transfection competent complexes were prepared by firstmixing 0.1 ug (per well) plasmid DNA (pCIluc-luciferase expressionplasmid) with 10 μl Opti-MEM reduced serum media, followed by sequentialaddition of the polycation and amphipathic compound. Ternary complexeswere incubated for 20 minutes before drop-wise addition to culturedcells. Cultured cells were grown in 100 μL of appropriate mediasupplemented with 10% FBS. No media change is requiredpost-transfection.

In this example EPEI served as the polycation and Bis-(N-MeHis)-ODAP (inFIG. 12 noted as 1161) served as the amphipathic compound. Comparisonswere made with several commercially available reagents using optimizedratios of reagent to DNA and following the manufacturer's recommendedprotocol (FIG. 12 ). Commercially available reagents included Fugene® HD(Promega), Lipofectamine® 2000 (Life Technologies) and Lipofectamine®LTX Plus (Life Technologies). Cells were harvested at 24 hours andassayed for luciferase activity. FIG. 12 depicts the mean relative lightunits for each experimental condition. The error bars represent thestandard deviation of the triplicate wells.

At optimal dose and ratio, EPEI+Bis-(N-MeHis)-ODAP resulted in up to36-fold increase in relative light units versus commercially availablereagents. This demonstrates the transfection efficiency ofEPEI+Bis-(N-MeHis)-ODAP when complexed with pDNA and transfected intocells in culture.

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

We claim:
 1. A composition for delivery of a nucleic acid into a cellcomprising: (i) a polymer comprising a first cyclic amine; and (ii) anamphipathic compound comprising a second cyclic amine; wherein saidpolymer is different from said amphipathic compound.
 2. The compositionof claim 1, wherein said first cyclic amine comprises (1) two or morecarbons and (2) at least one secondary or tertiary amine.
 3. Thecomposition of claim 1, wherein said first cyclic amine comprises astructure of:

wherein: R₂ and R₃ are each (CH₂)_(x), wherein each x is independently1, 2, 3, 4, 5, or 6; Y is N or CH; and R₄ is H or CH₃.
 4. Thecomposition of claim 3, wherein each x is independently 1, 2, or 3; andR⁴ is H.
 5. The composition of claim 1, wherein said polymer comprises apolyacrylate or a polyacrylamide.
 6. The composition of claim 1, whereinsaid polymer comprises 2 to about 80 monomeric units.
 7. The compositionof claim 1, wherein said polymer comprises greater than 80 monomericunits.
 8. The composition of claim 1, wherein said polymer comprises acopolymer.
 9. The composition of claim 8, wherein said first cyclicamine comprises a 5- to 7-membered cyclic amine.
 10. The composition ofclaim 8, wherein said copolymer comprises a first monomeric unitcomprising said first cyclic amine, and a second monomeric unitcomprising at least one secondary or tertiary amine.
 11. The compositionof claim 10, wherein said second monomeric unit comprises a structureof:

wherein: R₅ is H or CH₃, R₆ is an alkyl group containing one or moreprimary, secondary, or tertiary amines, and Z is NH or O.
 12. Thecomposition of claim 8, wherein said copolymer is a block copolymer, analternating copolymer, a random or statistical copolymer, or a gradientcopolymer.
 13. The composition of claim 8, wherein said copolymer has alinear structure, a graft structure, a branched structure, a crosslinkedor network structure, a star structure, a comb structure, a ladderstructure, or a dendritic structure.
 14. The composition of claim 1,wherein said second cyclic amine comprises a structure of:

wherein n is 0 or
 1. 15. The composition of claim 1, further comprisingsaid nucleic acid molecule.
 16. The composition of claim 15, whereinsaid nucleic acid molecule comprises DNA, RNA, or any combinationthereof.
 17. The composition of claim 16, wherein said RNA comprisesmRNA.
 18. A method of delivering a nucleic acid molecule into a cell,comprising: (a) providing a composition comprising: (i) a polymercomprising a cyclic amine; (ii) an amphipathic compound; and (iii) saidnucleic acid molecule; and (b) bringing said composition in contact withsaid cell, thereby delivering said nucleic acid molecule into said cell.19. The method of claim 18, wherein said nucleic acid molecule comprisesDNA or RNA.
 20. The method of claim 18, wherein said nucleic acidmolecule is delivered into said cell in vitro or in vivo.